Anti-EPHB4 antibodies and methods using same

ABSTRACT

The invention provides anti-EphB4 antibodies, and compositions comprising and methods of using these antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 12/160,084, filed Jan. 4, 2007 (Int'l), now U.S.Pat. No. 8,101,365, which is a national stage patent application ofPCT/US2007/060115, filed Jan. 4, 2007, which claims priority under 35USC §119 to U.S. Provisional Application No. 60/756,889, filed Jan. 5,2006, and U.S. Provisional Application No. 60/760,892, filed Jan. 20,2006, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology. More specifically, the invention concerns anti-EphB4antibodies, and uses of same.

BACKGROUND OF THE INVENTION

Development of a vascular supply is a fundamental requirement for manyphysiological and pathological processes. Actively growing tissues suchas embryos and tumors require adequate blood supply. They satisfy thisneed by producing pro-angiogenic factors, which promote new blood vesselformation via a process called angiogenesis. Vascular tube formation isa complex but orderly biological event involving all or many of thefollowing steps: a) endothelial cells (ECs) proliferate from existingECs or differentiate from progenitor cells; b) ECs migrate and coalesceto form cord-like structures; c) vascular cords then undergotubulogenesis to form vessels with a central lumen; d) existing cords orvessels send out sprouts to form secondary vessels; e) primitivevascular plexus undergo further remodeling and reshaping; and f)peri-endothelial cells are recruited to encase the endothelial tubes,providing maintenance and modulatory functions to the vessels; suchcells including pericytes for small capillaries, smooth muscle cells forlarger vessels, and myocardial cells in the heart. Hanahan, Science277:48-50 (1997); Hogan & Kolodziej, Nat. Rev. Genet. 3:513-23 (2002);Lubarsky & Krasnow, Cell 112:19-28 (2003).

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors andmetastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,chronic inflammation, intraocular neovascular diseases such asproliferative retinopathies, e.g., diabetic retinopathy, age-relatedmacular degeneration (AMD), neovascular glaucoma, immune rejection oftransplanted corneal tissue and other tissues, rheumatoid arthritis, andpsoriasis. Folkman et al., J. Biol. Chem. 267:10931-34 (1992); Klagsbrunet al., Annu. Rev. Physiol. 53:217-39 (1991); and Garner A., “Vasculardiseases,” In: Pathobiology of Ocular Disease. A Dynamic Approach,Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker, NY, 1994),pp 1625-1710.

In the case of tumor growth, angiogenesis appears to be crucial for thetransition from hyperplasia to neoplasia, and for providing nourishmentfor the growth and metastasis of the tumor. Folkman et al., Nature339:58 (1989). The neovascularization allows the tumor cells to acquirea growth advantage and proliferative autonomy compared to the normalcells. A tumor usually begins as a single aberrant cell which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. Accordingly, a correlation hasbeen observed between density of microvessels in tumor sections andpatient survival in breast cancer as well as in several other tumors.Weidner et al., N. Engl. J. Med. 324:1-6 (1991); Horak et al., Lancet340:1120-24 (1992); Macchiarini et al., Lancet 340:145-46 (1992). Theprecise mechanisms that control the angiogenic switch is not wellunderstood, but it is believed that neovascularization of tumor massresults from the net balance of a multitude of angiogenesis stimulatorsand inhibitors (Folkman, Nat. Med. 1(1):27-31 (1995)).

The process of vascular development is tightly regulated. To date, asignificant number of molecules, mostly secreted factors produced bysurrounding cells, have been shown to regulate EC differentiation,proliferation, migration and coalescence into cord-like structures. Forexample, vascular endothelial growth factor (VEGF) has been identifiedas the key factor involved in stimulating angiogenesis and in inducingvascular permeability. Ferrara et al., Endocr. Rev. 18:4-25 (1997). Thefinding that the loss of even a single VEGF allele results in embryoniclethality points to an irreplaceable role played by this factor in thedevelopment and differentiation of the vascular system. Furthermore,VEGF has been shown to be a key mediator of neovascularizationassociated with tumors and intraocular disorders. Ferrara et al.,Endocr. Rev. supra. The VEGF mRNA is overexpressed by the majority ofhuman tumors examined. Berkman et al., J. Clin. Invest. 91:153-59(1993); Brown et al., Human Pathol. 26:86-91 (1995); Brown et al.,Cancer Res. 53:4727-35 (1993); Mattern et al., Brit. J. Cancer 73:931-34(1996); Dvorak et al., Am. J. Pathol. 146:1029-39 (1995).

Also, the concentration levels of VEGF in eye fluids are highlycorrelated to the presence of active proliferation of blood vessels inpatients with diabetic and other ischemia-related retinopathies. Aielloet al., N. Engl. J. Med. 331:1480-87 (1994). Furthermore, studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD. Lopez et al., Invest. Ophthalmol. Vis. Sci.37:855-68 (1996).

Anti-VEGF neutralizing antibodies suppress the growth of a variety ofhuman tumor cell lines in nude mice (Kim et al., Nature 362:841-44(1993); Warren et al., J. Clin. Invest. 95:1789-97 (1995); Borgström etal., Cancer Res. 56:4032-39 (1996); Melnyk et al., Cancer Res. 56:921-24(1996)) and also inhibit intraocular angiogenesis in models of ischemicretinal disorders (Adamis et al., Arch. Ophthalmol. 114:66-71 (1996)).Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGFaction are promising candidates for the treatment of tumors and variousintraocular neovascular disorders. Such antibodies are described, forexample, in EP 817,648, published Jan. 14, 1998; and in WO 98/45331 andWO 98/45332, both published Oct. 15, 1998. One anti-VEGF antibody,bevacizumab, has been approved by the FDA for use in combination with achemotherapy regimen to treat metastatic colorectal cancer (CRC). Andbevacizumab is being investigated in many ongoing clinical trials fortreating various cancer indications.

The EphB4 receptor (“EphB4” or “EphB4R”) is a member of the eph receptorfamily, which constitutes the largest family of tyrosine kinasereceptors in the human genome (reviewed in Dodelet, Oncogene, 19:5614-5619, 2000). The human eph receptor tyrosine kinases arecategorized by sequence identity into an A class and a B class withcorresponding A-type and B-type ligands referred to as ephrins.Signaling can occur in a forward manner, in which the receptor tyrosinekinase is activated by the ligand, and in a reverse manner, in which thetransmembrane ephrinB ligands are activated by interaction withreceptors. Eph receptor ligand interactions have been implicated in awide range of biological functions including axon guidance, tissueborder formation, vasculogenesis, and cell motility (Kullander et al.Nat. Rev. Mol. Cell. Biol., 3: 475-486, 2002; Cheng et al. CytokineGrowth Factor Rev., 13: 75-85, 2002; Coulthard et al. Int. J. Dev.Biol., 46: 375-384, 2002). EphB4 binds ligands such as ephrin-B1,ephrin-B2, and ephrin-B3. The EphB4 receptor has an extracellular regionwith a cysteine-rich motif extending over its amino-terminal halffollowed by two fibronectin type II motifs. There is an intracellulardomain featuring a conserved kinase region and a transmembrane domain.

It is clear that there continues to be a need for agents that haveclinical attributes that are optimal for development as therapeuticagents. The invention described herein meets this need and providesother benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention is in part based on the identification of a variety ofEphB4 binding agents (such as immunoconjugates, antibodies, andfragments thereof). EphB4 presents as an important and advantageoustherapeutic target, and the invention provides compositions and methodsbased on binding EphB4. EphB4 binding agents of the invention, asdescribed herein, provide important therapeutic and diagnostic agentsfor use in targeting pathological conditions associated with expressionand/or activity of the EphB4-EphB4 ligand pathways. Accordingly, theinvention provides methods, compositions, kits and articles ofmanufacture related to EphB4 binding.

The present invention provides antibodies that bind (such asspecifically bind) to EphB4.

In one aspect, the invention provides an isolated anti-EphB4 antibody,wherein a full length IgG form of the antibody specifically binds humanEphB4 with a binding affinity of about 50 pM or better. As iswell-established in the art, binding affinity of a ligand to itsreceptor can be determined using any of a variety of assays, andexpressed in terms of a variety of quantitative values. Accordingly, inone embodiment, the binding affinity is expressed as Kd values andreflects intrinsic binding affinity (e.g., with minimized avidityeffects). Generally and preferably, binding affinity is measured invitro, whether in a cell-free or cell-associated setting. Any of anumber of assays known in the art, including those described herein, canbe used to obtain binding affinity measurements, including, for example,Biacore™, radioimmunoassay (RIA) and ELISA.

In one aspect, the invention provides an isolated antibody that binds aligand binding region of EphB4. In some embodiments, the isolatedantibody binds a polypeptide comprising, consisting of or consistingessentially of the EphB4 extracellular domain.

In one aspect, the invention provides an isolated anti-EphB4 antibodythat competes with EphB4 ligand for binding of EphB4.

In one aspect, the invention provides an isolated anti-EphB4 antibodythat inhibits, reduces, and/or blocks EphB4 activity. In someembodiments, EphB4 autophosphorylation is inhibited, reduced, and/orblocked.

In one aspect, an anti-EphB4 antibody of the invention comprises:

(a) at least one, two, three, four or five hypervariable region (HVR)sequences selected from the group consisting of:

(i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is RASQDVSTAVA(SEQ ID NO: 9)

(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is SASFLYS (SEQ IDNO:11)

(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QESTTTPPT (SEQID NO:15)

(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GFSISNYYLH(SEQ ID NO:2)

(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 isGGIYLYGSSSEYADSVKG (SEQ ID NO:4) and

(vi) HVR-H3 comprising sequence F1-F17, wherein F1-F17 isARGSGLRLGGLDYAMDY (SEQ ID NO:7); and

(b) at least one variant HVR, wherein the variant HVR sequence comprisesmodification of at least one residue of the sequence depicted in SEQ IDNOs:1-17.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or six HVRs, wherein each HVR comprises, consists orconsists essentially of a sequence selected from the group consisting ofSEQ ID NOs: 1-17, and wherein SEQ ID NO:9 or 10 correspond to an HVR-L1,SEQ ID NO:11 or 12 correspond to an HVR-L2, SEQ ID NO:13, 14, 15, 16, or17 correspond to an HVR-L3, SEQ ID NO:1 or 2 correspond to an HVR-H1,SEQ ID NO:3, 4, 5, or 6 correspond to an HVR-H2, and SEQ ID NO:7 or 8correspond to an HVR-H3.

In one embodiment, an antibody of the invention comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,comprises SEQ ID NO:9, 11, 13, 1, 3, 7.

In one embodiment, an antibody of the invention comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,comprises SEQ ID NO:10, 12, 14, 1, 3, 8.

In one embodiment, an antibody of the invention comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,comprises SEQ ID NO:9, 11, 15, 2, 4, 7.

In one embodiment, an antibody of the invention comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,comprises SEQ ID NO:9, 11, 16, 1, 5, 7.

In one embodiment, an antibody of the invention comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order,comprises SEQ ID NO:9, 11, 17, 1, 6, 7.

Variant HVRs in an antibody of the invention can have modifications ofone or more (such as two, three, four, five, or more) residues withinthe HVR.

In one embodiment, a HVR-L1 variant comprises 1-5 (1, 2, 3, 4, or 5)substitutions in any combination of the following positions: A6 (V orS), A7 (S or E), A8 (T or I), A9 (A or F), and A10 (V or L).

In one embodiment, a HVR-L2 variant comprises 1-2 (1, or 2)substitutions in any combination of the following positions: B4 (F or N)and B6 (Y or E).

In one embodiment, a HVR-L3 variant comprises 1-7 (1, 2, 3, 4, 5, 6 or7) substitutions in any combination of the following positions: C2 (Q,E, or K), C3 (S or T), C4 (Y, N, T, E, or A), C5 (T, A, or Q), C6 (T, V,or I), C8 (P, L, or E), and C9 (T or S).

In one embodiment, a HVR-H1 variant comprises 1-3 (1, 2, or 3)substitutions in any combination of the following positions: D3 (T orS), D6 (G or N), and D9 (I or L).

In one embodiment, a HVR-H2 variant comprises 1-5 (1, 2, 3, 4 or 5)substitutions in any combination of the following positions: E5 (P, orL), E7 (S or G), E8 (G or S), E10 (T, S, or R), and E11 (D, E, or G).

In one embodiment, a HVR-113 variant comprises 1 substitution in thefollowing positions: F3 (G or S). Letter(s) in parenthesis followingeach position indicates an illustrative substitution (i.e., replacement)amino acid; as would be evident to one skilled in the art, suitabilityof other amino acids as substitution amino acids in the contextdescribed herein can be routinely assessed using techniques known in theart and/or described herein. In one aspect, the invention provides anantibody comprising a HVR-H1 region comprising the sequence of SEQ IDNO:1 or 2. In one aspect, the invention provides an antibody comprisinga HVR-H2 region comprising the sequence of SEQ ID NO:3, 4, 5, or 6. Inone aspect, the invention provides an antibody comprising a HVR-H3region comprising the sequence of SEQ ID NO: 7 or 8. In one embodiment,the invention provides an antibody comprising a HVR-L1 region comprisingthe sequence of SEQ ID NO:9 or 10. In one embodiment, the inventionprovides an antibody comprising a HVR-L2 region comprising the sequenceof SEQ ID NO: 11 or 12. In one embodiment, the invention provides anantibody comprising a HVR-L3 region comprising the sequence of SEQ IDNO: 13, 14, 15, 16, or 17.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a HVR-H1 sequence comprising the sequence of SEQ ID NO:2;

(ii) a HVR-H2 sequence comprising the sequence of SEQ ID NO: 4;

(iii) a HVR-H3 sequence comprising the sequence of SEQ ID NO: 7.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three of the following:

(i) a HVR-L1 sequence comprising the sequence of SEQ ID NO: 9;

(ii) a HVR-L2 sequence comprising the sequence of SEQ ID NO: 11;

(iii) a HVR-L3 sequence comprising the sequence of SEQ ID NO: 15.

The amino acid sequences of SEQ ID NOs:1-17 are numbered with respect toindividual HVR (i.e., H1, H2 or H3) as indicated in FIG. 1, thenumbering being consistent with the Kabat numbering system as describedbelow.

In one aspect, the invention provides antibodies comprising heavy chainHVR sequences as depicted in FIG. 1.

In one aspect, the invention provides antibodies comprising light-chainHVR sequences as depicted in FIG. 1.

Some embodiments of antibodies of the invention comprise a light chainvariable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®,Genentech, Inc., South San Francisco, Calif., USA) (also referred to inU.S. Pat. No. 6,407,213 and Lee et al., J. Mol. Biol. (2004),340(5):1073-93) as depicted in SEQ ID NO:18 below.

-   -   1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val        Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr        Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu        Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser        Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln        Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro        Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (SEQ ID        NO: 18) (HVR residues are underlined)        In one embodiment, the huMAb4D5-8 light chain variable domain        sequence is modified at one or more of positions 30, 66 and 91        (Asn, Arg and His as indicated in bold/italics above,        respectively). In one embodiment, the modified huMAb4D5-8        sequence comprises Ser in position 30, Gly in position 66 and/or        Ser in position 91. Accordingly, in one embodiment, an antibody        of the invention comprises a light chain variable domain        comprising the sequence depicted in SEQ ID NO:52 below:

(SEQ ID NO: 52) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(HVR residues are underlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

Antibodies of the invention can comprise any suitable framework variabledomain sequence, provided binding activity to EphB4 is substantiallyretained. For example, in some embodiments, antibodies of the inventioncomprise a human subgroup III heavy chain framework consensus sequence.In one embodiment of these antibodies, the framework consensus sequencecomprises substitution at position 71, 73 and/or 78. In some embodimentsof these antibodies, position 71 is A, 73 is T and/or 78 is A. In oneembodiment, these antibodies comprise heavy chain variable domainframework sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., SouthSan Francisco, Calif., USA) (also referred to in U.S. Pat. Nos.6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol. (2004),340(5):1073-93). In one embodiment, these antibodies further comprise ahuman κI light chain framework consensus sequence. In one embodiment,these antibodies comprise light chain HVR sequences of huMAb4D5-8 asdescribed in U.S. Pat. Nos. 6,407,213 & 5,821,337.) In one embodiment,these antibodies comprise light chain variable domain sequences ofhuMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South San Francisco, Calif.,USA) (also referred to in U.S. Pat. Nos. 6,407,213 & 5,821,337, and Leeet al., J. Mol. Biol. (2004), 340(5):1073-93).

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, and/or 37, and HVR H1, H2 and H3 sequences are SEQ IDNOS:9, 11, and/or 15, respectively. In one embodiment, an antibody ofthe invention comprises a light chain variable domain, wherein theframework sequence comprise the sequence of SEQ ID NOS: 38, 39, 40and/or 41, and HVR L1, L2 and L3 sequences are SEQ ID NOS: 2, 4, and/or7, respectively.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NOS: 46, 47, 48, and/or 49, and HVR H1, H2 and H3 sequencesare SEQ ID NOS:2, 4, and/or 7, respectively. In one embodiment, anantibody of the invention comprises a light chain variable domain,wherein the framework sequence comprise the sequence of SEQ ID NOS: 42,43, 44, and/or 45, and HVR L1, L2 and L3 sequences are SEQ ID NOS:9, 11,and/or 15, respectively.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequencesof SEQ ID NOS: 46, 47, 51, and 49, and HVR H1, H2 and H3 sequences areSEQ ID NOS:2, 4, and/or 7, respectively. In one embodiment, an antibodyof the invention comprises a light chain variable domain, wherein theframework sequence comprise the sequences of SEQ ID NOS: 42, 43, 50, and45, and HVR L1, L2 and L3 sequences are SEQ ID NOS:9, 11 and/or 15,respectively.

In one embodiment, an antibody of the invention is affinity matured toobtain the target binding affinity desired. In one example, an affinitymatured antibody of the invention comprises substitution at one or moreof amino acid position H28, H31, H34, H52a, H54, H55, H57, H58, H95,L29, L30, L31, L32, L33, L53, L55, L90, L91, L92, L93, L94, L96, or L97.In one example an affinity matured antibody of the invention comprisesone or more of the following substitutions: (a) in the heavy chain,T28S, G31N, 134L, P52aL, S54G, G55S, T57S or R, D58, E or G, G95S, or(b), in the light chain, V29S, S30E, T31I, A32F, V33L, F53N, Y55E, Q90Eor K, S91T, Y92N T E or A, T93V or I, T94V or I, P96L or E, or T97S.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:53. In oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NO:54. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:53 and a lightchain variable domain comprising the sequence of SEQ ID NO:54.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:55. In oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NO:56. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:55 and a lightchain variable domain comprising the sequence of SEQ ID NO:56.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:57. In oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NO:58. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:57 and a lightchain variable domain comprising the sequence of SEQ ID NO:58.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:59. In oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NO:60. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:59 and a lightchain variable domain comprising the sequence of SEQ ID NO:60.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:61. In oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence of SEQ ID NO:62. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:61 and a lightchain variable domain comprising the sequence of SEQ ID NO:62.

In one aspect, the invention provides an antibody that competes with anyof the above-mentioned antibodies for binding to EphB4. In one aspect,the invention provides an antibody that binds to the same epitope onEphB4 as any of the above-mentioned antibodies.

As is known in the art, and as described in greater detail hereinbelow,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below).

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a polyclonal antibody. In some embodiments,the antibody is selected from the group consisting of a chimericantibody, an affinity matured antibody, a humanized antibody, and ahuman antibody. In some embodiments, the antibody is an antibodyfragment. In some embodiments, the antibody is a Fab, Fab′, Fab′-SH,F(ab)₂, or scFv.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human or humanized sequence (e.g.,framework and/or constant domain sequences). In one embodiment, thenon-human donor is a mouse. In one embodiment, an antigen bindingsequence is synthetic, e.g. obtained by mutagenesis (e.g., phage displayscreening, etc.). In one embodiment, a chimeric antibody of theinvention has murine V regions and human C region. In one embodiment,the murine light chain V region is fused to a human kappa light chain.In one embodiment, the murine heavy chain V region is fused to a humanIgG1 C region.

Humanized antibodies of the invention include those that have amino acidsubstitutions in the FR and affinity maturation variants with changes inthe grafted CDRs. The substituted amino acids in the CDR or FR are notlimited to those present in the donor or recipient antibody. In otherembodiments, the antibodies of the invention further comprise changes inamino acid residues in the Fc region that lead to improved effectorfunction including enhanced CDC and/or ADCC function and B-cell killing.Other antibodies of the invention include those having specific changesthat improve stability. In other embodiments, the antibodies of theinvention comprise changes in amino acid residues in the Fc region thatlead to decreased effector function, e.g. decreased CDC and/or ADCCfunction and/or decreased B-cell killing. In some embodiments, theantibodies of the invention are characterized by decreased binding (suchas absence of binding) to human complement factor C1q and/or human Fcreceptor on natural killer (NK) cells. In some embodiments, theantibodies of the invention are characterized by decreased binding (suchas the absence of binding) to human FcγRI, FcγRIIA, and/or FcγRIIIA. Insome embodiments, the antibodies of the invention is of the IgG class(eg, IgG1 or IgG4) and comprises at least one mutation in E233, L234,G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and/or P329(numbering according to the EU index). In some embodiments, theantibodies comprise the mutation L234A/L235A or D265A/N297A.

In one aspect, the invention provides anti-EphB4 polypeptides comprisingany of the antigen binding sequences provided herein, wherein theanti-EphB4 polypeptides specifically bind to EphB4.

The antibodies of the invention bind (such as specifically bind) EphB4,and in some embodiments, may modulate one or more aspects ofEphB4-associated effects, including but not limited to EphB4 activation,EphB4 downstream molecular signaling, EphB4 ligand activation, EphB4ligand downstream molecular signaling, disruption of ligand (e.g.,ephrin-B1, ephrin-B2, and/or ephrin-B3) binding to EphB4, EphB4phosphorylation and/or EphB4 multimerization, and/or EphB4 ligandphosphorylation, and/or disruption of any biologically relevant EphB4and/or EphB4 ligand biological pathway, inhibition of angiogenesis,and/or treatment and/or prevention of a tumor, cell proliferativedisorder or a cancer; and/or treatment or prevention of a disorderassociated with EphB4 expression and/or activity (such as increasedEphB4 expression and/or activity). In some embodiments, the antibody ofthe invention specifically binds to EphB4. In some embodiments, theantibody specifically binds to the EphB4 extracellular domain (ECD). Insome embodiments, the antibody specifically binds to a polypeptideconsisting of or consisting essentially of the EphB4 extracellulardomain. In some embodiments, the antibody specifically binds EphB4 witha KD of about 50 pM or stronger. In some embodiments, the antibody ofthe invention reduces, inhibits, and/or blocks EphB4 activity in vivoand/or in vitro. In some embodiments, the antibody reduces, inhibitsand/or blocks EphB4 autophosphorylation. In some embodiments, theantibody competes for binding with EphB4 ligand (reduces and/or blocksEphB4 ligand binding to EphB4).

In one aspect, the invention provides use of an antibody of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the disorder is aneuropathy or neurodegenerative disease.

In one aspect, the invention provides use of an antibody of theinvention in the preparation of a medicament for the inhibition ofangiogenesis.

In one aspect, the invention provides compositions comprising one ormore antibodies of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In one aspect, the invention provides nucleic acids encoding ananti-EphB4 antibody of the invention.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides compositions comprising one ormore nucleic acid of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods of making an antibody ofthe invention. For example, the invention provides methods of making ananti-EphB4 antibody (which, as defined herein includes full length andfragments thereof) or immunoconjugate, said method comprising expressingin a suitable host cell a recombinant vector of the invention encodingsaid antibody (or fragment thereof), and recovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more anti-EphB4antibodies of the invention. In one embodiment, the compositioncomprises a nucleic acid of the invention. In one embodiment, acomposition comprising an antibody further comprises a carrier, which insome embodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (for e.g., the antibody) to a subject(such as instructions for any of the methods described herein).

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more anti-EphB4 antibodies ofthe invention; and a second container comprising a buffer. In oneembodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antibody further comprises acarver, which in some embodiments is pharmaceutically acceptable. In oneembodiment, a kit further comprises instructions for administering thecomposition (for e.g., the antibody) to a subject.

In one aspect, the invention provides use of an anti-EphB4 antibody ofthe invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a cancer, a tumor,and/or a cell proliferative disorder. In some embodiments, the disorderis a neuropathy or neurodegenerative disease. In some embodiments, thedisorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the disorder is aneuropathy or neurodegenerative disease. In some embodiments, thedisorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the disorder is aneuropathy or neurodegenerative disease. In some embodiments, thedisorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the disorder is aneuropathy or neurodegenerative disease. In some embodiments, thedisorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a cancer, a tumor,and/or a cell proliferative disorder. In some embodiments, the disorderis a neuropathy or neurodegenerative disease. In some embodiments, thedisorder is a pathological condition associated with angiogenesis.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disorder, such as a cancer, a tumor, and/or a cellproliferative disorder.

In some embodiments, the disorder is a neuropathy or neurodegenerativedisease. In some embodiments, the disorder is a pathological conditionassociated with angiogenesis.

The invention provides methods and compositions useful for modulatingdisease states associated with expression and/or activity of EphB4, suchas increased or decreased expression and/or activity, or undesiredexpression and/or activity.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder associated withincreased expression and/or activity of EphB4, the methods comprisingadministering an effective amount of an anti-EphB4 antibody to a subjectin need of such treatment.

In one aspect, the invention provides methods for killing a cell (suchas a cancer or tumor cell), the methods comprising administering aneffective amount of an anti-EphB4 antibody to a subject in need of suchtreatment.

In one aspect, the invention provides methods for reducing, inhibiting,blocking, or preventing growth of a tumor or cancer, the methodscomprising administering an effective amount of an anti-EphB4 antibodyto a subject in need of such treatment.

In one aspect, the invention provides methods for treating or preventinga neuropathy or neurodegenerative disease, or repairing a damaged nervecell, the methods comprising administering an effective amount of ananti-EphB4 antibody to a subject in need of such treatment.

In one aspect, the invention provides methods for promoting thedevelopment, proliferation, maintenance or regeneration of neurons, themethods comprising administering an effective amount of an anti-EphB4antibody to a subject in need of such treatment.

In one aspect, the invention provides methods for inhibitingangiogenesis comprising administering an effective amount of ananti-EphB4 antibody to a subject in need of such treatment.

In one aspect, the invention provides methods for treating apathological condition associated with angiogenesis comprisingadministering an effective amount of an anti-EphB4 antibody to a subjectin need of such treatment. In some embodiments, the pathologicalcondition associated with angiogenesis is a tumor, a cancer, and/or acell proliferative disorder. In some embodiments, the pathologicalcondition associated with angiogenesis is an intraocular neovasculardisease.

Methods of the invention can be used to affect any suitable pathologicalstate. Exemplary disorders are described herein, and include a cancerselected from the group consisting of small cell lung cancer,neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectalcancer (CRC), and hepatocellular carcinoma.

In one embodiment, a cell that is targeted in a method of the inventionis a cancer cell. For example, a cancer cell can be one selected fromthe group consisting of a breast cancer cell, a colorectal cancer cell,a lung cancer cell, a papillary carcinoma cell, a colon cancer cell, apancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell,a central nervous system cancer cell, an osteogenic sarcoma cell, arenal carcinoma cell, a hepatocellular carcinoma cell, a bladder cancercell, a gastric carcinoma cell, a head and neck squamous carcinoma cell,a melanoma cell, a leukemia cell, and a colon adenoma cell. In oneembodiment, a cell that is targeted in a method of the invention is ahyperproliferative and/or hyperplastic cell. In one embodiment, a cellthat is targeted in a method of the invention is a dysplastic cell. Inyet another embodiment, a cell that is targeted in a method of theinvention is a metastatic cell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (for e.g., a cancer cell) isexposed to radiation treatment or a chemotherapeutic agent.

In one aspect, the invention provides methods comprising administrationof an effective amount of an anti-EphB4 antibody in combination with aneffective amount of another therapeutic agent (such as ananti-angiogenesis agent). For example, an anti-EphB4 antibody(ies) areused in combinations with anti-cancer agent or an anti-angiogenic agentto treat various neoplastic or non-neoplastic conditions. In oneembodiment, the neoplastic or non-neoplastic condition is a pathologicalcondition associated with angiogenesis. In some embodiments, the othertherapeutic agent is an anti-angiogenic agent, an anti-neoplastic agent,and/or a chemotherapeutic agent.

The anti-EphB4 antibody can be administered serially or in combinationwith the other therapeutic agent that is effective for those purposes,either in the same composition or as separate compositions. Theadministration of the anti-EphB4 antibody and the other therapeuticagent (e.g., anti-cancer agent, anti-angiogenic agent) can be donesimultaneously, e.g., as a single composition or as two or more distinctcompositions, using the same or different administration routes.Alternatively, or additionally, the administration can be donesequentially, in any order. Alternatively, or additionally, the stepscan be performed as a combination of both sequentially andsimultaneously, in any order. In certain embodiments, intervals rangingfrom minutes to days, to weeks to months, can be present between theadministrations of the two or more compositions. For example, theanti-cancer agent may be administered first, followed by the anti-EphB4antibody. However, simultaneous administration or administration of theanti-EphB4 antibody first is also contemplated. Accordingly, in oneaspect, the invention provides methods comprising administration of ananti-EphB4 antibody, followed by administration of an anti-angiogenicagent (such as an anti-VEGF antibody, such as bevacizumab). In certainembodiments, intervals ranging from minutes to days, to weeks to months,can be present between the administrations of the two or morecompositions.

In certain aspects, the invention provides a method of treating adisorder (such as a tumor, a cancer, and/or a cell proliferativedisorder) by administering effective amounts of an anti-EphB4 antibodyand/or an angiogenesis inhibitor(s) and one or more chemotherapeuticagents. A variety of chemotherapeutic agents may be used in the combinedtreatment methods of the invention. An exemplary and non-limiting listof chemotherapeutic agents contemplated is provided herein under“Definitions.” The administration of the anti-EphB4 antibody and thechemotherapeutic agent can be done simultaneously, e.g., as a singlecomposition or as two or more distinct compositions, using the same ordifferent administration routes. Alternatively, or additionally, theadministration can be done sequentially, in any order. Alternatively, oradditionally, the steps can be performed as a combination of bothsequentially and simultaneously, in any order. In certain embodiments,intervals ranging from minutes to days, to weeks to months, can bepresent between the administrations of the two or more compositions. Forexample, the chemotherapeutic agent may be administered first, followedby the anti-EphB4 antibody. However, simultaneous administration oradministration of the anti-EphB4 antibody first is also contemplated.Accordingly, in one aspect, the invention provides methods comprisingadministration of an anti-EphB4 antibody, followed by administration ofa chemotherapeutic agent. In certain embodiments, intervals ranging fromminutes to days, to weeks to months, can be present between theadministrations of the two or more compositions.

In one aspect, the invention provides methods of enhancing efficacy ofan anti-angiogenic agent in a subject having a pathological conditionassociated with angiogenesis, comprising administering to the subject aneffective amount of an anti-EphB4 antibody in combination with theanti-angiogenic agent, thereby enhancing said anti-angiogenic agent'sinhibitory activity.

In another aspect, the invention provides methods for detection ofEphB4, the methods comprising detecting EphB4-anti-EphB4 antibodycomplex in the sample. The term “detection” as used herein includesqualitative and/or quantitative detection (measuring levels) with orwithout reference to a control.

In another aspect, the invention provides methods for diagnosing adisorder associated with EphB4 expression and/or activity, the methodscomprising detecting EphB4-anti-EphB4 antibody complex in a biologicalsample from a patient having or suspected of having the disorder. Insome embodiments, the EphB4 expression is increased expression orabnormal expression. In some embodiments, the disorder is a tumor,cancer, and/or a cell proliferative disorder.

In another aspect, the invention provides any of the anti-EphB4antibodies described herein, wherein the anti-EphB4 antibody comprises adetectable label.

In another aspect, the invention provides a complex of any of theanti-EphB4 antibodies described herein and EphB4. In some embodiments,the complex is in vivo or in vitro. In some embodiments, the complexcomprises a cancer cell. In some embodiments, the anti-EphB4 antibody isdetectably labeled.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Heavy chain and light chain HVR loop sequences of anti-EphB4antibodies. The figure shows the heavy chain HVR sequences, H1, H2, andH3, and light chain HVR sequences, L1, L2 and L3. Sequence numbering isas follows: clone 30.35 (HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ ID NO:3;HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11;HVR-L3 is SEQ ID NO:13); clone 30.35.1D2 (HVR-H1 is SEQ ID NO:1; HVR-H2is SEQ ID NO:3; HVR-H3 is SEQ ID NO:8; HVR-L1 is SEQ ID NO:10; HVR-L2 isSEQ ID NO:12; HVR-L3 is SEQ ID NO:14); clone 30.35.2D8 (HVR-H1 is SEQ IDNO:2; HVR-H2 is SEQ ID NO:4; HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ IDNO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ ID NO:15); clone 30.35.2D12(HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ ID NO:5; HVR-H3 is SEQ ID NO:7;HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ ID NO:16);and clone 30.35.2D13 (HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ ID NO:6;HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11;HVR-L3 is SEQ ID NO:17).

Amino acid positions are numbered according to the Kabat numberingsystem as described below.

FIGS. 2A and 2B & 3 depict exemplary acceptor human consensus frameworksequences for use in practicing the instant invention with sequenceidentifiers as follows:

Variable Heavy (VH) Consensus Frameworks (FIGS. 2A and 2B)

human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NO:19)

human VH subgroup I consensus framework minus extended hypervariableregions (SEQ ID NOs:20-22)

human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NO:23)

human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOs:24-26)

human VH subgroup III consensus framework minus Kabat CDRs (SEQ IDNO:27)

human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NOs:28-30)

human VH acceptor framework minus Kabat CDRs (SEQ ID NO:31)

human VH acceptor framework minus extended hypervariable regions (SEQ IDNOs:32-33)

human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO:34)

human VH acceptor 2 framework minus extended hypervariable regions (SEQID NOs:35-37)

Variable Light (VL) Consensus Frameworks (FIG. 3)

human VL kappa subgroup I consensus framework (SEQ ID NO:38)

human VL kappa subgroup II consensus framework (SEQ ID NO:39)

human VL kappa subgroup III consensus framework (SEQ ID NO:40)

human VL kappa subgroup IV consensus framework (SEQ ID NO:41)

FIG. 4 depicts framework region sequences of huMAb4D5-8 light and heavychains. Numbers in superscript/bold indicate amino acid positionsaccording to Kabat.

FIG. 5 depicts modified/variant framework region sequences of huMAb4D5-8light and heavy chains. Numbers in superscript/bold indicate amino acidpositions according to Kabat.

FIG. 6 depicts the heavy chain variable regions and light chain variableregions of antibody clones 30.35, 30.35.1D2, 30.35.2D8, 30.35.2D12, and20.25.2D13.

FIG. 7: An anti-EphB4 monoclonal antibody blocked EphB4 receptorsignaling in a cell-based assay.

DETAILED DESCRIPTION OF THE INVENTION

The invention herein provides anti-EphB4 antibodies, that are usefulfor, e.g., treatment or prevention of disease states associated withexpression and/or activity of EphB4, such as increased expression and/oractivity or undesired expression and/or activity. In some embodiments,the antibodies of the invention are used to treat a tumor, a cancer,and/or a cell proliferative disorder.

In another aspect, the anti-EphB4 antibodies of the invention findutility as reagents for detection and/or isolation of EphB4, such asdetention of EphB4 in various tissues and cell type.

The invention further provides methods of making anti-EphB4 antibodies,and polynucleotides encoding anti-EphB4 antibodies.

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

DEFINITIONS

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol 293:865-881).To establish conditions for the assay, microtiter plates (Dynex™) arecoated overnight with 5 ug/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount™ gamma counter (Packard) for ten minutes. Concentrations ofeach Fab that give less than or equal to 20% of maximal binding arechosen for use in competitive binding assays. According to anotherembodiment the Kd or Kd value is measured by using surface plasmonresonance assays using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore,Inc., Piscataway, N.J.) at 25 C with immobilized antigen CM5 chips at^(˜)10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (−0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore™ Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) is calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. Mol.Biol 293:865-881. If the on-rate exceeds 10⁶M⁻¹S⁻¹ by the surfaceplasmon resonance assay above, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco™ spectrophotometer (ThermoSpectronic) with a stir redcuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CM5 chips at ^(˜)10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (−0.204) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcore™Evaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)was calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol 293:865-881. However, if the on-rate exceeds10⁶M⁻¹S⁻¹ by the surface plasmon resonance assay above, then the on-rateis preferably determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-Aminco™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table A below. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code shown in Table A below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. The ALIGN-2 program should be compiled for use ona UNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

The term “EphB4” (interchangeably termed “EphB4R”), as used herein,refers, unless specifically or contextually indicated otherwise, to anynative or variant (whether native or synthetic) EphB4 polypeptide. Theterm “native sequence” specifically encompasses naturally occurringtruncated or secreted forms (e.g., an extracellular domain sequence),naturally occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants. The term “wild type EphB4”generally refers to a polypeptide comprising the amino acid sequence ofa naturally occurring EphB4 protein. The term “wild type EphB4 sequence”generally refers to an amino acid sequence found in a naturallyoccurring EphB4.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL, dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In one embodiment, an antibody fragment comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcRn binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For e.g., such an antibodyfragment may comprise on antigen binding arm linked to an Fc sequencecapable of conferring in vivo stability to the fragment.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing, target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on pay 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or Presta U.S. Pat. No. 6,737,056 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIB (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëdron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins. WO00/42072 (Presta) describes antibody variants withimproved or diminished binding to FcRs. The content of that patentpublication is specifically incorporated herein by reference. See, also,Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the polypeptide or by recombinant engineering thenucleic acid encoding the polypeptide. Accordingly, a compositioncomprising a polypeptide having an Fc region according to this inventioncan comprise polypeptides with K447, with all K447 removed, or a mixtureof polypeptides with and without the K447 residue.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework, or from a human consensus framework.An acceptor human framework “derived from” a human immunoglobulinframework or human consensus framework may comprise the same amino acidsequence thereof, or may contain pre-existing amino acid sequencechanges. Where pre-existing amino acid changes are present, preferablyno more than 5 and preferably 4 or less, or 3 or less, pre-existingamino acid changes are present. Where pre-existing amino acid changesare present in a VH, preferably those changes are only at three, two orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may be 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences:

(SEQ ID NO: 46) EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 47)-H1-WVRQAPGKGLEWV (SEQ ID NO: 48) -H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC(SEQ ID NO: 49) -H3-WGQGTLVTVSS.In one embodiment, the VH subgroup III consensus framework amino acidsequence comprises RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:51).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 42) DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 43)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 44) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 45) -L3-FGQGTKVEIK.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; carcinoma, blastoma, and sarcoma.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and varioustypes of head and neck cancer. Dysregulation of angiogenesis can lead tomany disorders that can be treated by compositions and methods of theinvention. These disorders include both non-neoplastic and neoplasticconditions. Neoplastics include but are not limited those describedabove. Non-neoplastic disorders include but are not limited to undesiredor aberrant hypertrophy, arthritis, rheumatoid arthritis (RA),psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis,atherosclerotic plaques, diabetic and other proliferative retinopathiesincluding retinopathy of prematurity, retrolental fibroplasia,neovascular glaucoma, age-related macular degeneration, diabetic macularedema, corneal neovascularization, corneal graft neovascularization,corneal graft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, chronicinflammation, lung inflammation, acute lung injury/ARDS, sepsis, primarypulmonary hypertension, malignant pulmonary effusions, cerebral edema(e.g., associated with acute stroke/closed head injury/trauma), synovialinflammation, pannus formation in RA, myositis ossificans, hypertropicbone formation, osteoarthritis (OA), refractory ascites, polycysticovarian disease, endometriosis, 3rd spacing of fluid diseases(pancreatitis, compartment syndrome, burns, bowel disease), uterinefibroids, premature labor, chronic inflammation such as IBD (Crohn'sdisease and ulcerative colitis), renal allograft rejection, inflammatorybowel disease, nephrotic syndrome, undesired or aberrant tissue massgrowth (non-cancer), hemophilic joints, hypertrophic scars, inhibitionof hair growth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or disorder.

The terms “neurodegenerative disease” and “neurodegenerative disorder”are used in the broadest sense to include all disorders the pathology ofwhich involves neuronal degeneration and/or dysfunction, including,without limitation, peripheral neuropathies; motorneuron disorders, suchas amylotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Bell'spalsy, and various conditions involving spinal muscular atrophy orparalysis; and other human neurodegenerative diseases, such asAlzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis,Huntington's chorea, Down's Syndrome, nerve deafness, and Meniere'sdisease.

“Peripheral neuropathy” is a neurodegenerative disorder that affects theperipheral nerves, most often manifested as one or a combination ofmotor, sensory, sensorimotor, or autonomic dysfunction. Peripheralneuropathies may, for example, be genetically acquired, can result froma systemic disease, or can be induced by a toxic agent, such as aneurotoxic drug, e.g. antineoplastic agent, or industrial orenvironmental pollutant. “Peripheral sensory neuropathy” ischaracterized by the degeneration of peripheral sensory neurons, whichmay be idiopathic, may occur, for example, as a consequence of diabetes(diabetic neuropathy), cytostatic drug therapy in cancer (e.g. treatmentwith chemotherapeutic agents such as vincristine, cisplatin,methotrexate, 3′-azido-3′-deoxythymidine, or taxanes, e.g. paclitaxel[TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.] and doxetaxel[TAXOTERE®, Rhône-Poulenc Rorer, Antony, France]), alcoholism, acquiredimmunodeficiency syndrom (AIDS), or genetic predisposition. Geneticallyacquired peripheral neuropathies include, for example, Refsum's disease,Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease,Dejerine-Sottas syndrome, Abetalipoproteinemia, and Charcot-Marie-Tooth(CMT) Disease (also known as Proneal Muscular Atrophy or HereditaryMotor Sensory Neuropathy (HMSN)). Most types of periphseral neuropathydevelop slowly, over the course of several months or years. In clinicalpractice such neuropathies are called chronic. Sometimes a peripheralneuropathy develops rapidly, over the course of a few days, and isreferred to as acute. Peripheral neuropathy usually affects sensory andmotor nerves together so as to cause a mixed sensory and motorneuropathy, but pure sensory and pure motor neuropathy are also known.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows), sport animals, pets (such as cats, dogs and horses), primates,mice and rats.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®)); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA®zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingEphB4) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing EphB4) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent”. Examples of therapeutic agents (anti-cancer agents,also termed “anti-neoplastic agent” herein) include, but are limited to,e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxicagents, agents used in radiation therapy, anti-angiogenesis agents,apoptotic agents, anti-tubulin agents, toxins, and other-agents to treatcancer, e.g., anti-VEGF neutralizing antibody, VEGF antagonist,anti-HER-2, anti-CD20, an epidermal growth factor receptor (EGFR)antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor,erlotinib, a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe ErbB2, ErbB3, ErbB4, or VEGF receptor(s), inhibitors for receptortyrosine kinases for platet-derived growth factor (PDGF) and/or stemcell factor (SCF) (e.g., imatinib mesylate (Gleevec® Novartis)),TRAIL/Apo2, and other bioactive and organic chemical agents, etc.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Forexample, an anti-angiogenesis agent is an antibody or other antagonistto an angiogenic agent as defined above, e.g., antibodies to VEGF,antibodies to VEGF receptors, small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinibmalate), AMG706). Anti-angiogensis agents also include nativeangiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g.,Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit andDelmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing antiangiogenic factors); and,Sato Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 listsAnti-angiogenic agents used in clinical trials).

Compositions of the Invention and Methods of Making Same

This invention encompasses compositions, including pharmaceuticalcompositions, comprising an anti-EphB4 antibody; and polynucleotidescomprising sequences encoding an anti-EphB4 antibody. As used herein,compositions comprise one or more antibodies that bind to EphB4, and/orone or more polynucleotides comprising sequences encoding one or moreantibodies that bind to EphB4. These compositions may further comprisesuitable carriers, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

The invention also encompasses isolated antibody and polynucleotideembodiments. The invention also encompasses substantially pure antibodyand polynucleotide embodiments.

The anti-EphB4 antibodies of the invention are preferably monoclonal.Also encompassed within the scope of the invention are Fab, Fab′,Fab′-SH and F(ab′)₂ fragments of the anti-EphB4 antibodies providedherein. These antibody fragments can be created by traditional means,such as enzymatic digestion, or may be generated by recombinanttechniques. Such antibody fragments may be chimeric or humanized. Thesefragments are useful for the diagnostic and therapeutic purposes setforth below.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-EphB4 monoclonal antibodies of the invention can be made usingthe hybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to EphB4 generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of EphB4 and an adjuvant. EphB4 may be prepared using methodswell-known in the art, some of which are further described herein. Forexample, recombinant production of EphB4 is described below. In oneembodiment, animals are immunized with a derivative of EphB4 thatcontains the extracellular domain (ECD) of EphB4 fused to the Fc portionof an immunoglobulin heavy chain. In a preferred embodiment, animals areimmunized with an EphB4-IgG1 fusion protein. Animals ordinarily areimmunized against immunogenic conjugates or derivatives of EphB4 withmonophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (RibiImmunochem. Research, Inc., Hamilton, Mont.) and the solution isinjected intradermally at multiple sites. Two weeks later the animalsare boosted. 7 to 14 days later animals are bled and the serum isassayed for anti-EphB4 titer. Animals are boosted until titer plateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against EphB4. Preferably,the binding specificity of monoclonal antibodies produced by hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbentassay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The anti-EphB4 antibodies of the invention can be made by usingcombinatorial libraries to screen for synthetic antibody clones with thedesired activity or activities. In principle, synthetic antibody clonesare selected by screening phage libraries containing phage that displayvarious fragments of antibody variable region (Fv) fused to phage coatprotein. Such phage libraries are panned by affinity chromatographyagainst the desired antigen. Clones expressing Fv fragments capable ofbinding to the desired antigen are adsorbed to the antigen and thusseparated from the non-binding clones in the library. The binding clonesare then eluted from the antigen, and can be further enriched byadditional cycles of antigen adsorption/elution. Any of the anti-EphB4antibodies of the invention can be obtained by designing a suitableantigen screening procedure to select for the phage clone of interestfollowed by construction of a full length anti-EphB4 antibody cloneusing the Fv sequences from the phage clone of interest and suitableconstant region (Fc) sequences described in Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-EphB4 clones is desired, the subject is immunized withEphB4 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-EphB4 clones isobtained by generating an anti-EphB4 antibody response in transgenicmice carrying a functional human immunoglobulin gene array (and lackinga functional endogenous antibody production system) such that EphB4immunization gives rise to B cells producing human antibodies againstEphB4. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-EphB4 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing EphB4-specific membrane bound antibody, e.g., by cellseparation with EphB4 affinity chromatography or adsorption of cells tofluorochrome-labeled EphB4 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which EphB4 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

EphB4 nucleic acid and amino acid sequences are known in the art.Nucleic acid sequence encoding the EphB4 can be designed using the aminoacid sequence of the desired region of EphB4. Alternatively, the cDNAsequence (or fragments thereof) of GenBank™ Accession Nos. NM_(—)004444,or disclosed in U.S. Pat. No. 5,635,177, can be used. DNAs encodingEphB4 can be prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, chemical synthesis by any ofthe methods described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989), such as the triester, phosphite, phosphoramidite andH-phosphonate methods. In one embodiment, codons preferred by theexpression host cell are used in the design of the EphB4 encoding DNA.Alternatively, DNA encoding the EphB4 can be isolated from a genomic orcDNA library.

Following construction of the DNA molecule encoding the EphB4, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. In general,plasmid vectors contain replication and control sequences which arederived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells. Suitable vectors for expression in prokaryotic andeukaryotic host cells are known in the art and some are thitherdescribed herein. Eukaryotic organisms, such as yeasts, or cells derivedfrom multicellular organisms, such as mammals, may be used.

Optionally, the DNA encoding the EphB4 is operably linked to a secretoryleader sequence resulting in secretion of the expression product by thehost cell into the culture medium. Examples of secretory leadersequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephosphatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Methods fortransfection are well known in the art, and some are further describedherein.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Methods fortransformation are well known in the art, and some are further describedherein.

Prokaryotic host cells used to produce the EphB4 can be cultured asdescribed generally in Sambrook et al., supra.

The mammalian host cells used to produce the EphB4 can be cultured in avariety of media, which is well known in the art and some of which isdescribed herein.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Purification of EphB4 may be accomplished using art-recognized methods,some of which are described herein.

The purified EphB4 can be attached to a suitable matrix such as agarosebeads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the EphB4 protein to the matrix can be accomplished by themethods described in Methods in Enzymology, vol. 44 (1976). A commonlyemployed technique for attaching protein ligands to polysaccharidematrices, e.g. agarose, dextran or cellulose, involves activation of thecarrier with cyanogen halides and subsequent coupling of the peptideligand's primary aliphatic or aromatic amines to the activated matrix.

Alternatively, EphB4 can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other art-known method for panning phage displaylibraries.

The phage library samples are contacted with immobilized EphB4 underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88:3978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by EphB4 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1.000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for EphB4.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting EphB4, rare high affinity phage could becompeted out. To retain all the higher affinity mutants, phages can beincubated with excess biotinylated EphB4, but with the biotinylatedEphB4 at a concentration of lower molarity than the target molaraffinity constant for EphB4. The high affinity-binding phages can thenbe captured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics.

Anti-EphB4 cones may be activity selected. In one embodiment, theinvention provides anti-EphB4 antibodies that block the binding betweenan EphB4 ligand (such as ephrin-B1, ephrin-B2 and/or ephrin-B3) andEphB4, but do not block the binding between an EphB4 ligand and a secondprotein (such as EphB1, EphB3, EphB4, EphB5 and/or EphB6). Fv clonescorresponding to such anti-EphB4 antibodies can be selected by (1)isolating anti-EphB4 clones from a phage library as described above, andoptionally amplifying the isolated population of phage clones by growingup the population in a suitable bacterial host; (2) selecting EphB4 anda second protein against which blocking and non-blocking activity,respectively, is desired; (3) adsorbing the anti-EphB4 phage clones toimmobilized EphB4; (4) using an excess of the second protein to eluteany undesired clones that recognize EphB4-binding determinants whichoverlap or are shared with the binding determinants of the secondprotein; and (5) eluting the clones which remain adsorbed following step(4). Optionally, clones with the desired blocking/non-blockingproperties can be further enriched by repeating the selection proceduresdescribed herein one or more times.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones of the invention is readily isolated and sequencedusing conventional procedures (e.g. by using oligonucleotide primersdesigned to specifically amplify the heavy and light chain codingregions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Reviewarticles on recombinant expression in bacteria of antibody-encoding DNAinclude Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) andPluckthun, Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding anti-EphB4 antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂, fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Human Antibodies

Human anti-EphB4 antibodies of the invention can be constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalanti-EphB4 antibodies of the invention can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forEphB4 and the other is for any other antigen. Exemplary bispecificantibodies may bind to two different epitopes of the EphB4 protein.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express EphB4. These antibodies possess an EphB4-binding armand an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fe region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc,wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: asp, glu;    -   (4) basic: his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine. In accordance with this description and the teachings ofthe art, it is contemplated that in some embodiments, an antibody usedin methods of the invention may comprise one or more alterations ascompared to the wild type counterpart antibody, e.g. in the Fc region.These antibodies would nonetheless retain substantially the samecharacteristics required for therapeutic utility as compared to theirwild type counterpart. For example, it is thought that certainalterations can be made in the Fc region that would result in altered(i.e., either improved or diminished) C1q binding and/or ComplementDependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See alsoDuncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S.Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fcregion variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman)describe antibody variants with improved or diminished binding to FcRs.The content of these patent publications are specifically incorporatedherein by reference. See, also, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half lives and improvedbinding to the neonatal Fc receptor (FcRn), which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are describedin US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fcregion with one or more substitutions therein which improve binding ofthe Fc region to FcRn. Polypeptide variants with altered Fc region aminoacid sequences and increased or decreased C1q binding capability aredescribed in U.S. Pat. No. 6,194,551B1, WO99/51642. The contents ofthose patent publications are specifically incorporated herein byreference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.Screening for Antibodies with Desired Properties

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art. In some embodiments, antibodies are characterized forany one or more of reduction or blocking of EphB4 activation, reductionor blocking of EphB4 downstream molecular signaling, reduction orblocking of EphB4 ligand activation, reduction or blocking or EphB4ligand downstream molecular signaling, disruption or blocking of ligand(e.g., ephrin-B1, ephrin-B2, and/or ephrin-B3) binding to EphB4, EphB4phosphorylation and/or EphB4 multimerization, and/or EphB4 ligandphosphorylation, and/or treatment and/or prevention of a tumor, cellproliferative disorder or a cancer; and/or treatment or prevention of adisorder associated with EphB4 expression and/or activity (such asincreased EphB4 expression and/or activity).

The purified antibodies can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. Illustrative antigen binding assay are providedbelow in the Examples section.

In still another embodiment, the invention provides anti-EphB4monoclonal antibodies that compete with 30.35, 30.35.1D2 and/or30.35.2D8 antibody for binding to EphB4. Such competitor antibodiesinclude antibodies that recognize an EphB4 epitope that is the same asor overlaps with the EphB4 epitope recognized by antibody 30.35,30.35.1D2 and/or 30.35.2D8. Such competitor antibodies can be obtainedby screening anti-EphB4 hybridoma supernatants for binding toimmobilized EphB4 in competition with labeled 30.35, 30.35.1D2 and/or30.35.2D8 antibody. A hybridoma supernatant containing competitorantibody will reduce the amount of bound, labeled antibody detected inthe subject competition binding mixture as compared to the amount ofbound, labeled antibody detected in a control binding mixture containingirrelevant (or no) antibody. Any of the competition binding assaysdescribed herein are suitable for use in the foregoing procedure.

In another aspect, the invention provides an anti-EphB4 monoclonalantibody that comprises one or more (such as 2, 3, 4, 5, and/or 6) HVRsof the 30.35, 30.35.1D2 or 30.35.2D8 antibody. An anti-EphB4 monoclonalantibody that comprises one or more HVR(s) of 30.35, 30.35.1D2 and/or30.35.2D8 can be constructed by grafting one or more HVR(s) of 30.35,30.35.1D2 and/or 30.35.2D8 onto a template antibody sequence, e.g. ahuman antibody sequence which is closest to the corresponding murinesequence of the parental antibody, or a consensus sequence of all humanantibodies in the particular subgroup of the parental antibody light orheavy chain, and expressing the resulting chimeric light and/or heavychain variable region sequence(s), with or without accompanying constantregion sequence(s), in recombinant host cells as described herein.

Anti-EphB4 antibodies of the invention possessing the unique propertiesdescribed herein can be obtained by screening anti-EphB4 hybridomaclones for the desired properties by any convenient method. For example,if an anti-EphB4 monoclonal antibody that blocks or does not block thebinding of EphB4 ligands to EphB4 is desired, the candidate antibody canbe tested in a binding competition assay, such as a competitive bindingELISA, wherein plate wells are coated with EphB4, and a solution ofantibody in an excess of the Eph ligand of interest is layered onto thecoated plates, and bound antibody is detected enzymatically, e.g.contacting the bound antibody with HRP-conjugated anti-Ig antibody orbiotinylated anti-Ig antibody and developing the HRP color reaction,e.g. by developing plates with streptavidin-HRP and/or hydrogen peroxideand detecting the HRP color reaction by spectrophotometry at 490 nm withan ELISA plate reader.

If an anti-EphB4 antibody that inhibits or activates EphB4 activation isdesired, the candidate antibody can be testing an EphB4 phosphorylationassay. Such assays are known in the art and one such assay is describedin the Examples section.

If an anti-EphB4 antibody inhibits cell growth is desired, the candidateantibody can be tested in in vitro and/or in vivo assays that measureinhibition of cell growth. Such assays are known in the art and arefurther described and exemplified herein.

In one embodiment, the present invention contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried outto confirm that the antibody is unable to bind C1q and hence lacks CDCactivity. To assess complement activation, a CDC assay, e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art,e.g. those described in the Examples section.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coliλ 1776 (ATCC31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex™ G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon™ or Millipore™ Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the anti-OX40Lantibodies described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (eg., above). Enzymatically active toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC 1065, and the derivatives of these toxins thathave toxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) of the invention conjugated to one or moremaytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹¹³, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

v. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody. Additional methods for preparing ADC are described herein.Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl)aminobenzoate (“STAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the present invention may be used in, for example, invitro, ex vivo and in vivo therapeutic methods.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder associated withincreased expression and/or activity of EphB4, the methods comprisingadministering an effective amount of an anti-EphB4 antibody to a subjectin need of such treatment.

In one aspect, the invention provides methods for reducing, inhibiting,or preventing growth of a tumor or cancer, the methods comprisingadministering an effective amount of an anti-EphB4 antibody to a subjectin need of such treatment.

EphB4 has been implicated in motoaxon guidance and neural crest cellmigration in the developing embryo. Accordingly, the antibodies of theinvention are also useful in the treatment (including prevention) ofdisorders the pathology of which involves cellular degeneration ordisfunction, such as treatment of various (chronic) neurodegenerativedisorders and acute nerve cell injuries. Such neurodegenerativedisorders include, without limitation, peripheral neuropathies;motorneuron disorders, such as amylotrophic lateral schlerosis (ALS, LouGehrig's disease), Bell's palsy, and various conditions involving spinalmuscular atrophy or paralysis; and other human neurodegenerativediseases, such as Alzheimer's disease, Parkinson's disease, epilepsy,multiple schlerosis, Huntington's chorea, Down's Syndrome, nervedeafness, and Meniere's disease, and acute nerve cell injuries, forexample due to trauma or spinal cord injury.

The antibodies of the invention are also useful for inhibitingangiogenesis. In some embodiments, the site of angiogenesis is a tumoror cancer.

In one aspect, the invention provides methods for inhibitingangiogenesis comprising administering an effective amount of ananti-EphB4 antibody to a subject in need of such treatment.

In one aspect, the invention provides methods for treating apathological condition associated with angiogenesis comprisingadministering an effective amount of an anti-EphB4 antibody to a subjectin need of such treatment. In some embodiments, the pathologicalcondition associated with angiogenesis is a tumor, a cancer, and/or acell proliferative disorder. In some embodiments, the pathologicalcondition associated with angiogenesis is an intraocular neovasculardisease.

Moreover, at least some of the antibodies of the invention can bindantigen from other species. Accordingly, the antibodies of the inventioncan be used to bind specific antigen activity, e.g., in a cell culturecontaining the antigen, in human subjects or in other mammalian subjectshaving the antigen with which an antibody of the invention cross-reacts(e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig ormouse). In one embodiment, the antibody of the invention can be used forinhibiting antigen activities by contacting the antibody with theantigen such that antigen activity is inhibited. Preferably, the antigenis a human protein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor binding an antigen in a subject suffering from a disorder associatedwith increased antigen expression and/or activity, comprisingadministering to the subject an antibody of the invention such that theantigen in the subject is bound. Preferably, the antigen is a humanprotein molecule and the subject is a human subject. Alternatively, thesubject can be a mammal expressing the antigen with which an antibody ofthe invention binds. Still further the subject can be a mammal intowhich the antigen has been introduced (e.g., by administration of theantigen or by expression of an antigen transgene). An antibody of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, an antibody of the invention can be administered toa non-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration).

The antibodies of the invention can be used to treat, inhibit, delayprogression of, prevent/delay recurrence of, ameliorate, or preventdiseases, disorders or conditions associated with expression and/oractivity of one or more antigen molecules.

Exemplary disorders include carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, cancer of the urinary tract, hepatoma, breast cancer, coloncancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, melanoma, multiple myeloma and B-celllymphoma, brain, as well as head and neck cancer, and associatedmetastases. In some embodiments, the cancer is selected from the groupconsisting of small cell lung cancer, neuroblastomas, melanoma, breastcarcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellularcarcinoma.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with one or more cytotoxic agent(s) is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell. In oneembodiment, the cytotoxic agent targets or interferes with microtubulepolymerization. Examples of such cytotoxic agents include any of thechemotherapeutic agents noted herein (such as a maytansinoid,auristatin, dolastatin, or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, chemotherapeuticagent(s) (including cocktails of chemotherapeutic agents), othercytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growthinhibitory agent(s). Where an antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth.Alternatively, or additionally, the patient may receive combinedradiation therapy (e.g. external beam irradiation or therapy with aradioactive labeled agent, such as an antibody). Such combined therapiesnoted above include combined administration (where the two or moreagents are included in the same or separate formulations), and separateadministration, in which case, administration of the antibody of theinvention can occur prior to, and/or following, administration of theadjunct therapy or therapies.

Combination Therapies

As indicated above, the invention provides combined therapies in whichan anti-EphB4 antibody is administered with another therapy. Forexample, anti-EphB4 antibodies are used in combinations with anti-cancertherapeutics or an anti-neovascularization therapeutics to treat variousneoplastic or non-neoplastic conditions. In one embodiment, theneoplastic or non-neoplastic condition is characterized by pathologicaldisorder associated with aberrant or undesired angiogenesis. Theanti-EphB4 antibody can be administered serially or in combination withanother agent that is effective for those purposes, either in the samecomposition or as separate compositions. Alternatively, or additionally,multiple inhibitors of EphB4 can be administered.

The administration of the anti-EphB4 antibody can be donesimultaneously, e.g., as a single composition or as two or more distinctcompositions using the same or different administration routes.Alternatively, or additionally, the administration can be donesequentially, in any order. In certain embodiments, intervals rangingfrom minutes to days, to weeks to months, can be present between theadministrations of the two or more compositions. For example, theanti-cancer agent may be administered first, followed by the EphB4inhibitor. However, simultaneous administration or administration of theanti-EphB4 antibody first is also contemplated.

The effective amounts of therapeutic agents administered in combinationwith an anti-EphB4 antibody will be at the physician's or veterinarian'sdiscretion. Dosage administration and adjustment is done to achievemaximal management of the conditions to be treated. The dose willadditionally depend on such factors as the type of therapeutic agent tobe used and the specific patient being treated. Suitable dosages for theanti-cancer agent are those presently used and can be lowered due to thecombined action (synergy) of the anti-cancer agent and the anti-EphB4antibody. In certain embodiments, the combination of the inhibitorspotentiates the efficacy of a single inhibitor. The term “potentiate”refers to an improvement in the efficacy of a therapeutic agent at itscommon or approved dose.

Typically, the anti-EphB4 antibodies and anti-cancer agents are suitablefor the same or similar diseases to block or reduce a pathologicaldisorder such as tumor growth or growth of a cancer cell. In oneembodiment the anti-cancer agent is an anti-angiogenesis agent.

Antiangiogenic therapy in relationship to cancer is a cancer treatmentstrategy aimed at inhibiting the development of tumor blood vesselsrequired for providing nutrients to support tumor growth. Becauseangiogenesis is involved in both primary tumor growth and metastasis,the antiangiogenic treatment provided by the invention is capable ofinhibiting the neoplastic growth of tumor at the primary site as well aspreventing metastasis of tumors at the secondary sites, thereforeallowing attack of the tumors by other therapeutics.

Many anti-angiogenic agents have been identified and are known in thearts, including those listed herein, e.g., listed under Definitions, andby, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al.,Nature Reviews: Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin.Oncol., 8:200-206 (2003). See also, US Patent Application US20030055006.In one embodiment, an anti-EphB4 antibody is used in combination with ananti-VEGF neutralizing antibody (or fragment) and/or another VEGFantagonist or a VEGF receptor antagonist including, but not limited to,for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3,neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of blockingVEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weightinhibitors of VEGFR tyrosine kinases (RTK), antisense strategies forVEGF, ribozymes against VEGF or VEGF receptors, antagonist variants ofVEGF; and any combinations thereof. Alternatively, or additionally, twoor more angiogenesis inhibitors may optionally be co-administered to thepatient in addition to VEGF antagonist and other agent. In certainembodiment, one or more additional therapeutic agents, e.g., anti-canceragents, can be administered in combination with anti-EphB4 antibody, theVEGF antagonist, and an anti-angiogenesis agent.

In certain aspects of the invention, other therapeutic agents useful forcombination tumor therapy with a anti-EphB4 antibody include othercancer therapies, (e.g., surgery, radiological treatments (e.g.,involving irradiation or administration of radioactive substances),chemotherapy, treatment with anti-cancer agents listed herein and knownin the art, or combinations thereof). Alternatively, or additionally,two or more antibodies binding the same or two or more differentantigens disclosed herein can be co-administered to the patient.Sometimes, it may be beneficial to also administer one or more cytokinesto the patient.

Chemotherapeutic Agents

In certain aspects, the invention provides a method of blocking orreducing tumor growth or growth of a cancer cell, by administeringeffective amounts of an antagonist of EphB4 and/or an angiogenesisinhibitor(s) and one or more chemotherapeutic agents to a patientsusceptible to, or diagnosed with, cancer. A variety of chemotherapeuticagents may be used in the combined treatment methods of the invention.An exemplary and non-limiting list of chemotherapeutic agentscontemplated is provided herein under “Definitions.”

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. Variation in dosage will likely occur depending onthe condition being treated. The physician administering treatment willbe able to determine the appropriate dose for the individual subject.

Relapse Tumor Growth

The invention also provides methods and compositions for inhibiting orpreventing relapse tumor growth or relapse cancer cell growth. Relapsetumor growth or relapse cancer cell growth is used to describe acondition in which patients undergoing or treated with one or morecurrently available therapies (e.g., cancer therapies, such aschemotherapy, radiation therapy, surgery, hormonal therapy and/orbiological therapy/immunotherapy, anti-VEGF antibody therapy,particularly a standard therapeutic regimen for the particular cancer)is not clinically adequate to treat the patients or the patients are nolonger receiving any beneficial effect from the therapy such that thesepatients need additional effective therapy. As used herein, the phrasecan also refer to a condition of the “non-responsive/refractory”patient, e.g., which describe patients who respond to therapy yet sufferfrom side effects, develop resistance, do not respond to the therapy, donot respond satisfactorily to the therapy, etc. In various embodiments,a cancer is relapse tumor growth or relapse cancer cell growth where thenumber of cancer cells has not been significantly reduced, or hasincreased, or tumor size has not been significantly reduced, or hasincreased, or fails any further reduction in size or in number of cancercells. The determination of whether the cancer cells are relapse tumorgrowth or relapse cancer cell growth can be made either in vivo or invitro by any method known in the art for assaying the effectiveness oftreatment on cancer cells, using the art-accepted meanings of “relapse”or “refractory” or “non-responsive” in such a context. A tumor resistantto anti-VEGF treatment is an example of a relapse tumor growth.

The invention provides methods of blocking or reducing relapse tumorgrowth or relapse cancer cell growth in a subject by administering oneor more anti-EphB4 antibody to block or reduce the relapse tumor growthor relapse cancer cell growth in subject. In certain embodiments, theantagonist can be administered subsequent to the cancer therapeutic. Incertain embodiments, the anti-EphB4 antibody are administeredsimultaneously with cancer therapy. Alternatively, or additionally, theanti-EphB4 antibody therapy alternates with another cancer therapy,which can be performed in any order. The invention also encompassesmethods for administering one or more inhibitory antibodies to preventthe onset or recurrence of cancer in patients predisposed to havingcancer. Generally, the subject was or is concurrently undergoing cancertherapy. In one embodiment, the cancer therapy is treatment with ananti-angiogenesis agent, e.g., a VEGF antagonist. The anti-angiogenesisagent includes those known in the art and those found under theDefinitions herein. In one embodiment, the anti-angiogenesis agent is ananti-VEGF neutralizing antibody or fragment (e.g., humanized A4.6.1,AVASTIN® (Genentech, South San Francisco, Calif.), Y0317, M4, G6, B20,2C3, etc.). See, e.g., U.S. Pat. Nos. 6,582,959, 6,884,879, 6,703,020;WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; US PatentApplications 20030206899, 20030190317, 20030203409, and 20050112126;Popkov et al., Journal of Immunological Methods 288; 149-164 (2004);and, WO2005012359. Additional agents can be administered in combinationwith VEGF antagonist and an anti-EphB4 antibody for blocking or reducingrelapse tumor growth or relapse cancer cell growth, e.g., see sectionentitled Combination Therapies herein.

The antibody of the invention (and adjunct therapeutic agent) is/areadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, e.g. by injections, suchas intravenous or subcutaneous injections, depending in part on whetherthe administration is brief or chronic.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents such as chemotherapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. One exemplary dosage of theantibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, e.g. about six doses of the antibody). An initial higher loadingdose, followed by one or more lower doses may be administered. Anexemplary dosing regimen comprises administering an initial loading doseof about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kgof the antibody. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

The anti-EphB4 antibodies of the invention are useful in assaysdetecting EphB4 expression (such as diagnostic or prognostic assays) inspecific cells or tissues wherein the antibodies are labeled asdescribed below and/or are immobilized on an insoluble matrix.

In another aspect, the invention provides methods for detection ofEphB4, the methods comprising detecting EphB4-anti-EphB4 antibodycomplex in the sample. The term “detection” as used herein includesqualitative and/or quantitative detection (measuring levels) with orwithout reference to a control.

In another aspect, the invention provides methods for diagnosing adisorder associated with EphB4 expression and/or activity, the methodscomprising detecting EphB4-anti-EphB4 antibody complex in a biologicalsample from a patient having or suspected of having the disorder. Insome embodiments, the EphB4 expression is increased expression orabnormal (undesired) expression. In some embodiments, the disorder is atumor, cancer, and/or a cell proliferative disorder.

In another aspect, the invention provides any of the anti-EphB4antibodies described herein, wherein the anti-EphB4 antibody comprises adetectable label.

In another aspect, the invention provides a complex of any of theanti-EphB4 antibodies described herein and EphB4. In some embodiments,the complex is in vivo or in vitro. In some embodiments, the complexcomprises a cancer cell. In some embodiments, the anti-EphB4 antibody isdetectably labeled.

Anti-EphB4 antibodies can be used for the detection of EphB4 in any oneof a number of well known detection assay methods. For example, abiological sample may be assayed for EphB4 by obtaining the sample froma desired source, admixing the sample with anti-EphB4 antibody to allowthe antibody to form antibody/EphB4 complex with any EphB4 present inthe mixture, and detecting any antibody/EphB4 complex present in themixture. The biological sample may be prepared for assay by methodsknown in the art which are suitable for the particular sample. Themethods of admixing the sample with antibodies and the methods ofdetecting antibody/EphB4 complex are chosen according to the type ofassay used. Such assays include immunohistochemistry, competitive andsandwich assays, and steric inhibition assays.

Analytical methods for EphB4 all use one or more of the followingreagents: labeled EphB4 analogue, immobilized EphB4 analogue, labeledanti-EphB4 antibody, immobilized anti-EphB4 antibody and stericconjugates. The labeled reagents also are known as “tracers.”

The label used is any detectable functionality that does not interferewith the binding of EphB4 and anti-EphB4 antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels include:The label used is any detectable functionality that does not interferewith the binding of EphB4 and anti-EphB4 antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol.Methods, 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem.,30: 407-412 (1982). Preferred labels herein are enzymes such ashorseradish peroxidase and alkaline phosphatase. The conjugation of suchlabel, including the enzymes, to the antibody is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the anti-EphB4 antibody from any EphB4that remains free in solution. This conventionally is accomplished byeither insolubilizing the anti-EphB4 antibody or EphB4 analogue beforethe assay procedure, as by adsorption to a water-insoluble matrix orsurface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling(for example, using glutaraldehyde cross-linking), or by insolubilizingthe anti-EphB4 antibody or EphB4 analogue afterward, e.g., byimmunoprecipitation.

The expression of proteins in a sample may be examined usingimmunohistochemistry and staining protocols. Immunohistochemicalstaining of tissue sections has been shown to be a reliable method ofassessing or detecting presence of proteins in a sample.Immunohistochemistry (“IHC”) techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods. For sample preparation, a tissue or cell samplefrom a mammal (typically a human patient) may be used. Examples ofsamples include, but are not limited to, cancer cells such as colon,breast, prostate, ovary, lung, stomach, pancreas, lymphoma, and leukemiacancer cells. The sample can be obtained by a variety of proceduresknown in the art including, but not limited to surgical excision,aspiration or biopsy. The tissue may be fresh or frozen. In oneembodiment, the sample is fixed and embedded in paraffin or the like.The tissue sample may be fixed (i.e. preserved) by conventionalmethodology. One of ordinary skill in the art will appreciate that thechoice of a fixative is determined by the purpose for which the sampleis to be histologically stained or otherwise analyzed. One of ordinaryskill in the art will also appreciate that the length of fixationdepends upon the size of the tissue sample and the fixative used.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigen(e.g., EphB4) is determined directly. This direct assay uses a labeledreagent, such as a fluorescent tag or an enzyme-labeled primaryantibody, which can be visualized without further antibody interaction.In a typical indirect assay, unconjugated primary antibody binds to theantigen and then a labeled secondary antibody binds to the primaryantibody. Where the secondary antibody is conjugated to an enzymaticlabel, a chromogenic or fluorogenic substrate is added to providevisualization of the antigen. Signal amplification occurs becauseseveral secondary antibodies may react with different epitopes on theprimary antibody.

The primary and/or secondary antibody used for immunohistochemistrytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired. For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out (see, e.g., Leong atal. Appl. Immunohistochem. 4(3):201 (1996)).

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation. The extent of binding ofantibody to the sample is determined by using any one of the detectablelabels discussed above. Preferably, the label is an enzymatic label(e.g. HRPO) which catalyzes a chemical alteration of the chromogenicsubstrate such as 3,3′-diaminobenzidine chromogen. Preferably theenzymatic label is conjugated to antibody which binds specifically tothe primary antibody (e.g. the primary antibody is rabbit polyclonalantibody and secondary antibody is goat anti-rabbit antibody).

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope, and stainingintensity criteria, routinely used in the art, may be employed. Stainingintensity criteria may be evaluated as follows:

TABLE 2 Staining Pattern Score No staining is observed in cells. 0Faint/barely perceptible staining is detected in more than 10% 1+ of thecells. Weak to moderate staining is observed in more than 10% of the 2+cells. Moderate to strong staining is observed in more than 10% of 3+the cells.

Typically, a staining pattern score of about 2+ or higher in an IHCassay is diagnostic and/or prognostic. In some embodiments, a stainingpattern score of about 1+ or higher is diagnostic and/or prognostic. Inother embodiments, a staining pattern score of about 3 of higher isdiagnostic and/or prognostic. It is understood that when cells and/ortissue from a tumor or colon adenoma are examined using IHC, staining isgenerally determined or assessed in tumor cell and/or tissue (as opposedto stromal or surrounding tissue that may be present in the sample).

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer EphB4 analogue tocompete with the test sample EphB4 for a limited number of anti-EphB4antibody antigen-binding sites. The anti-EphB4 antibody generally isinsolubilized before or after the competition and then the tracer andEphB4 bound to the anti-EphB4 antibody are separated from the unboundtracer and EphB4. This separation is accomplished by decanting (wherethe binding partner was preinsolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample EphB4 is inversely proportional to the amount ofbound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of EphB4 are prepared andcompared with the test results to quantitatively determine the amount ofEphB4 present in the test sample. These assays are called ELISA systemswhen enzymes are used as the detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theEphB4 is prepared and used such that when anti-EphB4 antibody binds tothe EphB4 the presence of the anti-EphB4 antibody modifies the enzymeactivity. In this case, the EphB4 or its immunologically activefragments are conjugated with a bifunctional organic bridge to an enzymesuch as peroxidase. Conjugates are selected for use with anti-EphB4antibody so that binding of the anti-EphB4 antibody inhibits orpotentiates the enzyme activity of the label. This method per se iswidely practiced under the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small EphB4 fragment so that antibodyto hapten is substantially unable to bind the conjugate at the same timeas anti-EphB4 antibody. Under this assay procedure the EphB4 present inthe test sample will bind anti-EphB4 antibody, thereby allowinganti-hapten to bind the conjugate, resulting in a change in thecharacter of the conjugate hapten, e.g., a change in fluorescence whenthe hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of EphB4or anti-EphB4 antibodies. In sequential sandwich assays an immobilizedanti-EphB4 antibody is used to adsorb test sample EphB4, the test sampleis removed as by washing, the bound EphB4 is used to adsorb a second,labeled anti-EphB4 antibody and bound material is then separated fromresidual tracer. The amount of bound tracer is directly proportional totest sample EphB4. In “simultaneous” sandwich assays the test sample isnot separated before adding the labeled anti-EphB4. A sequentialsandwich assay using an anti-EphB4 monoclonal antibody as one antibodyand a polyclonal anti-EphB4 antibody as the other is useful in testingsamples for EphB4.

The foregoing are merely exemplary detection assays for EphB4. Othermethods now or hereafter developed that use anti-EphB4 antibody for thedetermination of EphB4 are included within the scope hereof, includingthe bioassays described herein.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition(s)effective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody of the invention; and (b)a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat a particular condition, e.g. cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES Example 1 Generation of Anti-EphB4 Antibodies

Anti-EphB4 antibodies were generated using phage display, usingEphB4-His protein to select phage. A variety of methods are known in theart for generating phage display libraries from which an antibody ofinterest can be obtained. One method of generating antibodies ofinterest is through the use of a phage antibody library as described inLee et al., J. Mol. Biol. (2004), 340(5):1073-93. Clones selected usingphage display were screened against EphB4-His protein using phage ELISA.Unique clones were selected for further characterization by phagecompetition ELISA and a blocking assay to determine whether the phageantibody clone could block EphB4-ephrinB2 interactions. Clone 30.35performed favorably and was selected for further analysis. To improvethe affinity of clone 30.35, phage display libraries were generated inthe background of YW30.35, each targeting selected HVRs for soft or hardrandomization. Selected clones were screened by phage ELISA and thenexpressed as Fab protein and their affinity determined using Biacore.Selected clones were reformatted as full-length IgGs, and their affinitywas determined using Biacore™. The sequences of parent clone 30.35 andaffinity matured clones are shown in FIG. 1.

Example 2 Characterization of Anti-EphB4 Antibodies

To determine the binding affinity of anti-EphB4 Mabs, surface plasmonresonance (SRP) measurement with a BIAcore™-3000 was used (BIAcore,Inc., Piscataway, N.J.). Briefly, carboxymethylated dextran biosensorchips (CM5, BIAcore Inc.) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti-EphB4 antibody was diluted with 10 mM sodium acetate, pH 4.8, into5 ug/ml before injection at a flow rate of 5 ul/minute to achieveapproximately 500 response units (RU) of coupled antibody. Next, 1Methanolamine was injected to block unreacted groups. For kineticsmeasurements, two-fold serial dilutions of either human or murineEphB4-His molecules (0.7 nM to 500 nM) were injected in PBS with 0.05%Tween 20 at 25° C. at a flow rate of 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model (BIAcore™ Evaluation Software version3.2). The equilibrium dissociation constant (Kd) was calculated as theratio k_(off)/k_(on). The results of this experiment are shown in Table3. “NA” signifies that the measurement was not performed.

TABLE 3 Binding affinity and kinetics of anti-EphB4 antibodies to humanand mouse EphB4. murine antigen human antigen kon/ koff/ Kd kon/ koff/Kd Anti-EphB4 10⁵ 10⁻⁴ (nM) 10⁵ 10⁻⁴ (nM) YW30.35 0.31 0.75 2.4 0.030.96 32 YW30.35.1D2 1.28 <0.05 <0.04 0.36 <0.05 <0.14 YW30.35.2D8 0.92<0.05 <0.05 0.4 <0.05 <0.12 YW30.35.2D12 NA NA NA 0.27 <0.05 <0.19YM30.35.2D13 NA NA NA 0.45 <0.05 <0.11

In different experiments, the cross-reactivity of anti-EphB4 antibodyclone 30.35 was tested against other EphB receptors. Briefly, COS7 cellswere transiently transfected with plasmids expressing full-length humanEphB1, human EphB2, human EphB4 or human EphB6. 24 hours aftertransfection, cells were subjected to FACS analysis to detect binding byanti-EphB4 antibodies, if any. Anti-EphB4 clone 30.35 did notcross-react with human EphB1, human EphB2, human EphB3 or human EphB6.

Example 3 Anti-Eph4 Antibody Blocked EphB4 Receptor Signaling in aCell-Based Assay

In order to demonstrate the ability of anti-EphB4 antibodies to blockthe interaction of membrane-bound EphrinB2 and EphB4, we performed acell-based assay in which EphB4 and EphrinB2 were presented by differentcell types. 3T3 cells overexpressing human EphrinB2 were used tostimulate HUVEC cells that express high level of EphB4 but low level ofEphrinB2, and the ability of anti-EphB4 antibody to inhibit EphB4activation was tested.

3T3 cells overexpressing human EphrinB2 were prepared as follows: humanfull length EphrinB2 was cloned into pcDNA5/FRT vector (Invitrogen) andsubsequently used to generate stable cell line with 3T3.Flp cells(Invitrogen) according to manufacture's manual.

3T3 cells overexpressing human EphrinB2 were overlayed on HUVEC cellsfor 15 or 30 minutes, in the presence or absence of anti-EphB4 antibody.Activation of EphB4 receptor was assessed by immunoprecipitating EphB4protein, then detection of presence or absence of receptor tyrosinephosphorylation using an anti-phosphotyrosine antibody (antibody 4G10;Upstate) using western blotting. Briefly, cells were lysed with RIPAbuffer. Cell lysates were clarified by centrifugation and anti-EphB4antibody 35.2D8 was added at 5 ug/sample. After incubation at 4 C fortwo hours, the immunocomplex was pulled down using Protein A agarose.EphB4 phosphorylation was analyzed by Western Blot usinganti-phosphotyrosine antibody 4G10 (Upstate) at a concentration of 1ug/ml.

The results of this experiment are shown in FIG. 7. Overlay of 3T3 cellson HUVEC cells caused dramatic tyrosine phosphorylation of EphB4 (lane 4and 5). A 30 minute preincubation of HUVECs with anti-EphB4 antibody(clone 35.2D8 at 5 ug/ml) effectively abolished EphB4 tyrosinephosphorylation induced by overlaying 3T3-EphrinB2 cells (lane 6). Bycontrast, treatment of HUVEC cells with anti-EphB4 alone did not causeEphB4 activation in HUVECs (lane 2 and 3), and untreated HUVEC cells didnot demonstrate EphB4 activation. These results established thatanti-EphB4 antibody blocked ligand-receptor interaction in the contextof direct cell-cell contact.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

What is claimed is:
 1. A method of inhibiting angiogenesis comprisingadministration of an effective amount of an anti-EphB4 antibody to asubject in need of such treatment, wherein the anti-EphrinB4 antibodycomprises six hypervariable regions (HVRs) and wherein the antibodycomprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, whereineach, in order, comprises SEQ ID NO:9, 11, 15, 2, 4, and
 7. 2. Themethod of claim 1, further comprising administering to the subject aneffective amount of an anti-angiogenic agent.
 3. The method of claim 2,wherein the anti-angiogenic agent is administered prior to or subsequentto the administration of the anti-EphB4 antibody.
 4. The method of claim2, wherein the anti-angiogenic agent is administered concurrently withthe anti-EphB4 antibody.
 5. The method of claim 2, wherein theanti-angiogenic agent is an anti-VEGF antibody.
 6. The method of claim5, wherein the anti-VEGF antibody is AVASTIN® (bevacizumab).
 7. Themethod of claim 1, further comprising administering an effective amountof a chemotherapeutic agent.
 8. The method of claim 1, wherein theantibody comprises a human consensus framework sequence selected fromthe group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and
 41. 9. Themethod of claim 1, wherein the antibody comprises human κ subgroupconsensus framework sequence selected from the group consisting of SEQID NOs:38, 39, 40, and
 41. 10. The method of claim 1, wherein theantibody comprises a variable heavy (VH) subgroup III consensusframework sequence comprising at least a portion or all of: (a)EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:46); (b) WVRQAPGKGLEWV (SEQ IDNO:47); (c) RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO:48); and (d)WGQGTLVTVSS (SEQ ID NO:49).
 11. The method of claim 1, wherein theantibody comprises a VH subgroup III consensus framework sequencecomprising RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:51).
 12. The methodof claim 1, wherein the anti-EphB4 antibody is conjugated to adetectable label or cytotoxic agent.